1. Field of the Invention
The present invention relates generally to the field of viral vectors, packaging cell lines, and the use of such viral vectors to express foreign DNA in mammalian cells. The invention also relates to the field of gene therapy, and more specifically to the use of viral vectors to transport genetic material into cells in vivo for therapeutic purposes. More particularly, it concerns viral promoter replacement in order to reduce the expression levels of viral genes in host cells.
2. Description of the Related Art
Gene therapy is an area that offers an attractive approach for the treatment of many diseases and disorders. Many diseases are the result of genetic abnormalities such as gene mutations or deletions, and thus the prospect of replacing a damaged or missing gene with a fully functional gene is provocative. Throughout the last decade, studies of oncogenes and tumor suppressor genes have revealed increasing amounts of evidence that cancer is a disease caused by multiple genetic changes (Chiao et al., 1990; Levine, 1990; Weinberg, 1991; Sugimara et al., 1992). Based on this concept of carcinogenesis, new strategies of therapy have evolved rapidly as alternatives to conventional therapies such as chemo- and radiotherapy (Renan, 1990; Lotze et al., 1992; Pardoll, 1992). One of these strategies is gene therapy, in which tumor suppressor genes, antisense oligonucleotides, and other related genes are used to suppress the growth of malignant cells.
Gene therapy has also been contemplated for transfer of other therapeutically important genes into cells to correct genetic defects. Such genetic defects include deficiencies of adenosine deaminase that result in severe combined immunodeficiency, human blood clotting factor IX in hemophilia B, the dystrophin gene in Duchenne muscular dystrophy, and the cystic fibrosis transmembrane receptor in cystic fibrosis. Gene transfer in these situations requires long term expression of the transgene, and the ability to transfer large DNA fragments, such as the dystrophin cDNA, which is about 14 kB in size.
High efficiency transduction of cells and the ability to administer multiple doses of a therapeutic gene are particularly important points in gene therapy. The ability to transfer a gene into a cell requires a method of transferring the new genetic material across the plasma membrane of the cell and subsequent expression of the gene product to produce an effect on the cell. There are several means to transfer genetic material into a cell, including direct injection, lipofection, transfection of a plasmid, or transduction by a viral vector. The natural ability of viruses to infect a cell and direct gene expression make viral vectors attractive as gene transfer vectors. Other desirable elements of gene transfer vectors include a high transduction efficiency, large capacity for genetic material, targeted gene delivery, tissue-specific gene expression, and the ability to minimize host immunologic responses against the vector.
One particularly gene therapy vector, adenovirus, has been widely studied and well-characterized as a model system for eukaryotic gene expression, and have become the vector of choice for in vivo gene transfer. Adenoviruses are easy to grow and manipulate, and they exhibit broad host range both in vitro and in vivo. They can be produced to high titers, e.g., 109–1011 plaque-forming units (PFU)/ml, and they are highly infectious for both dividing and non-dividing cells. The life cycle of adenovirus does not require integration into the host cell genome; the foreign genes encoded by adenovirus vectors are expressed episomally, and therefore have low genotoxicity to the host cells. Adenoviruses are not, however, associated with any significant pathologies. They appear only to be linked to mild forms of disease, and there are no known human malignancies associated with adenovirus infection. Moreover, no side effects have, been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Many viral vectors have not showed the in vivo results that many have hoped, adenovirus being one of these. Expression levels and duration of expression appear to be two problems. It is thought that one of the causes for these problems is the toxicity and immunogenicity of adenovirus, especially and high dosage.
One way of attaining this goal is to reduce or eliminate the expression of adenoviral proteins in the host. The diminution of viral gene expression and viral replication is desirable for the development of viral vectors used for gene therapy, for attenuated live viral vaccines and for the transformation of cells in vitro for the purpose of protein production. A common approach to this endeavor in the adenoviral system has been to delete certain viral genes. Of course, if the gene is essential to viral replication, the function must be complemented. This complementation is accomplished by providing a “helper” cell line that is transformed with a copy of the deleted viral gene. When this cell is infected, the gene produces its essential product, thereby allowing the virus to replicate. However, in a cell not so transformed, the virus can infect but will not replicate.
Thus, there are many benefits to be obtained by the development of new viral vectors and methods for reducing the viral gene expression of such vectors in host cells.